Blood coagulation is a process consisting of complex interactions between various blood components (or factors) that eventually gives rise to a fibrin clot. Generally, the blood components that participate in what is frequently referred to as the coagulation “cascade” are enzymatically inactive proteins (proenzymes or zymogens) that are converted to proteolytic enzymes by the action of an activator, which itself is often an activated clotting factor. One peptide that is critical to the coagulation cascade is Factor VIII or FVIII. In fact, Hemophilia A, which is the most common hereditary coagulation disorder, is caused by deficiency or structural defects in Factor VIII. The biochemistry of Factor VIII allows for a rapid on/off switch for coagulation. It circulates as an inactive cofactor which is activated to FVIIIa by thrombin, the penultimate enzyme of the coagulation cascade. FVIIIa participates in a short-lived enzymatic complex (FXase) with FIXa, a membrane or phospholipid (PL) surface and Ca+2 to convert FX to FXa. The major function of FVIIIa as a participant in the FXase complex is to markedly amplify FXa, which then allows for thrombin generation. Factor VIII is encoded by a ˜186 kb gene consisting of 26 exons (Thompson, Seminars in Thrombosis and Hemostasis, 29:11-22 (2003) (references 11 and 16-18)). Translation of the mRNA of this gene, followed by removal of a 19 amino acid signal sequence, leads to a mature protein of 2332 amino acids. The protein consists of 6 major domains, which, from the amino terminus, are: A1, A2, B, A3, C1, and C2. Additional short acidic regions a1, a2, and a3, which are involved in activation, are interspersed between the A1 and A2, A2 and B, and B and A3 domains, respectively. The 2332 amino acid primary translation product is processed into a heterodimer consisting of a heterogeneous heavy chain, which contains the intact A1,a1 A2,a2 domains and various lengths of the B-domain, and a homogenous 80 kD light chain, which contains of a3, A3, C1, and C2 domain. The heterogeneity of the B-domain results from proteolysis during secretion.
Activation of Factor VIII to Factor VIIIa generally occurs via proteolysis of the procofactor by thrombin. Thrombin recognizes certain amino acid regions that define thrombin cleavage sites along the Factor VIII peptide chain. Factor VIII has three thrombin cleavage sites. Examination of other thrombin substrates reveals a variety of residues that can be accommodated by thrombin. Though the amino acids within these thrombin cleavage sites can vary to some degree, certain amino acids are much more common within these cleavage sites than others, and certain amino acid residues result in a more efficient cleavage of the peptide by thrombin. (See, e.g., Newell-Caito et al., “P3-P3′ Residues flanking Scissile Bonds in Factor VIII Modulate Rates of Substrate Cleavage and Profactor Activation by Thrombin,” Biochemistry 51:3451-59 (2012); Gallwitz et al., “The Extended Cleavage Specificity of Human Thrombin,” PLoS ONE 7:e31756 (2012)). One of the three thrombin cleavage sites in Factor VIII lies at or near the a1-A2 junction, which is at or near amino acid positions 370-375 of the mature wild-type human Factor VIII peptide.
Following cleavage, active Factor VIIIa is a heterotrimer comprised of the A1 subunit, the A2 subunit, and the A3C1C2 subunit. This heterotrimer is supported by both electrostatic and hydrophobic interactions between the subunits at the regions in which they interact with one another, termed the “domain interfaces.” The heterotrimer is known to include at least A1-A2 and A2-A3 domain interfaces. (See, e.g., U.S. Pat. No. 8,338,571 (filed Jul. 25, 2008) (issued Dec. 25, 2012)).
Human Factor VIII has been produced recombinantly as a single-chain molecule of approximately 300 kD. The precursor product is processed into two polypeptide chains of 200 kD (heavy) and 80 kD (light) in the Golgi Apparatus, with the two chains held together by metal ions (Kaufman et al., J. Biol. Chem. 263:6352 (1988); Andersson et al., Proc. Natl. Acad. Sci. 83:2979 (1986)). The B-domain of FVIII seems to be dispensable, as B-domain deleted FVIII (FVIII-BDD; 90 kD A1-A2 heavy chain plus 80 kD light chain) has also been shown to be effective as a replacement therapy for hemophilia A. One well-known B-domain deleted Factor VIII sequence referred to as “BDD-SQ” or simply “BDD” contains a deletion of all but 14 amino acids of the B-domain.
Treatment of hemophilia A currently involves intravenous (iv) administration of Factor VIII on demand or as a prophylactic therapy. Despite its large size of greater than 300 kD for the full-length protein, Factor VIII has a half-life in humans of only about 11 hours. (Ewenstein et al., Semin. Hematol. 41:1-16 (2004)). As such, Factor VIII must be administered relatively frequently for prophylactic treatment of clotting disorders. Factor VIII is typically administered two to three times a week with dosing based upon Factor VIII activity. This need for frequent iv injection creates tremendous barriers to patient compliance. It would be more convenient for patients if a Factor VIII product could be developed that required less frequent administration. Furthermore, reducing the number of dosages required would also reduce the cost of treatment. Additionally, even with these frequent administrations, due to its short half-life, patients undergoing Factor VIII replacement therapy often achieve large swings in plasma Factor VIII activity levels, potentially putting them at risk for thrombosis (at peak levels) and bleeding (at trough levels).
Additionally, an alternate, non-iv route of administration, such as subcutaneous (sc) administration, could both increase ease of treatment and decrease the potential risks of thrombosis and bleeding by maintaining plasma Factor VIII levels at a more constant level. One challenge of sc delivery is increasing the bioavailability of the administered Factor VIII. A Factor VIII peptide with enhanced activity could be useful in enhancing bioavailability, and therefore could be useful in sc delivery of Factor VIII. Due to its higher specific activity, such a Factor VIII peptide could allow for a decrease in the volume needed for administration, as the activity concentration of the drug is higher. Reduced injection volume could decrease patient discomfort. Furthermore, the reduction in volume would also translate to a reduction in the cost of goods. Finally, an enhanced activity Factor VIII molecule could also confer additional protection above that of wild-type Factor VIII, by prolonging the duration of cofactor activity if dosing is by mass rather than by activity. As an example, with equal mass dosing, the enhanced activity FVIII variants with 2-fold specific activity enhancement would offer the same protection at the 0.5% level as the protection provided at the 1% level for the wild-type FVIII, thus extending the interval between Factor VIII doses.
Numerous Factor VIII variants have been produced in an attempt to address one or more shortcomings of the current medical therapy. For example, U.S. Pat. No. 8,338,571 (filed Jul. 25, 2008; issued Dec. 25, 2012) describes a recombinant factor VIII that includes one or more mutations that result in enhanced stability of both Factor VIII and Factor VIIIa. Similarly, U.S. Patent Publication No. 2012/0190623 (filed Jan. 27, 2011) describes Factor VIII muteins that are resistant to inactivation, including muteins “wherein the APC cleavage sites, Arg336 and Ile562, are mutated.” U.S. Patent Publication No. 2011/0124565 (filed Apr. 10, 2006) relates to modified nucleic acid sequences coding for coagulation factors, in particular human Factor VIII and their derivatives with improved stability, including a Factor VIII peptide with a modification that reportedly prevents thrombin cleavage between the A1 and the A2 domain of FVIII. Other efforts have produced, for example, modified Factor VIII polypeptides that reportedly have increased circulating half-lives due to the introduction of mutations that permit PEGylation of the peptide (Mei et al., Blood 116:270-279 (2010)) and modified Factor VIII polypeptides that reportedly possess increased stability due to mutations to the amino acids that make up the domain interfaces of the active FVIIIa heterotrimer (Wakabayashi et al., J. Thromb. Haemost. 7:438-444 (2009)).
For the reasons stated above, there exists a need for improved Factor VIII variants, for instance a variant that possesses increased activity and/or a variant that need not be administered as frequently and/or at as high a dose. Furthermore, it is desirable that such a protein be produced as a homogeneous product in a consistent manner.